1. Field of the Invention
The present invention relates to a heterodimer protein composition that is efficiently and stably separated and purified.
2. Brief Description of the Background Art
Monoclonal antibodies are biological polymers having high binding affinity and specificity for antigens, and applied as molecular targeted therapeutics for the treatment of cancer, immune disorders or the like. The known action mechanisms of immunoglobulin G (hereinafter, abbreviated to IgG) which is widely used as a therapeutic monoclonal antibody include neutralization of antigens, receptor agonist or antagonist action, induction of apoptosis, phagocytosis, induction of complement or effector cell-dependent cytotoxicity, delivery of chemotherapeutic agents, toxins, and radioactive isotopes, or the like.
IgG antibodies are tetrameric molecules composed of two antibody heavy chains (hereinafter, abbreviated to H chain) and two antibody light chains (hereinafter, abbreviated to L chain), consisting of a variable region as an antigen-binding site and a constant region having a constant amino acid sequence (FIG. 1).
Each structure of an antibody is known to be involved in the various roles. The antigen-binding site is formed by a heavy chain variable region (hereinafter, abbreviated to VH) and a light chain variable region (hereinafter, abbreviated to VL), and various antigen specificities and binding affinities are caused by variability in primary sequence and higher-order structure.
Each constant region of IgG H chain (hereinafter, abbreviated to CH) is composed of CH1, hinge, CH2, and CH3 domains, and among them, hinge, CH2, and CH3 constitute the Fc region (hereinafter, also simply referred to as Fc). Fc binds to the Fcγ receptor (hereinafter, abbreviated to FcγR) present on leukocytes to be involved in release of inflammatory mediators, phagocytosis, and antibody-dependent cellular cytotoxicity (hereinafter, abbreviated to ADCC), and also binds to complement components to be involved in complement-dependent cytotoxicity (hereinafter, abbreviated to CDC).
Further, Fc of IgG antibody binds to the neonatal Fc receptor (hereinafter, abbreviated to FcRn) in the endosome under low pH conditions, and avoids lysosomal degradation to be involved in extension of the blood half-life. The primary sequence of Fc region varies depending on its subclasses (IgG1, IgG2, IgG3, IgG4), and its binding activity for FcγR, FcRn and complement components also differs, for this reason, they cause differences in biological properties (Non-Patent Document 1).
IgG antibody molecule has two fragments called as Fab, which is composed of VH, CH1, VL, and L chain constant region (hereinafter, abbreviated to CL), and these two Fabs form the bivalent binding with antigen molecules. This bivalent binding activity increases avidity for antigens, as well as induces intracellular signaling by cross-linking antigens when the antibodies bind to antigen molecules such as receptors present on the surface of cell membrane.
In order to avoid the bivalent antigen-binding activity of IgG antibody, antibody engineering consideration has been made, such as preparation of monovalent antibodies having a single antigen-binding site or antibody variants having an Fc variant containing substitution of amino acid residues for removing the FcγR binding activity. Meanwhile, Fab fragment including an antigen-binding site shows a monovalent binding activity for antigens, but shows a short blood half-life and is not useful as a therapeutic agent because there is no Fc (Non-Patent Document 2).
Meanwhile, bispecific antibodies are allowed to have the action mechanisms that cannot be achieved by the conventional IgG monoclonal antibodies, because of their bispecificity. For example, bispecific T cell engager [BiTE (registered trade name)] binds with both CD3 and a cancer antigen to recruit T cells and to efficiently damage cancer cells (Non-Patent Document 3). In addition, bispecific antibodies have a possibility of inhibiting activated intracellular signaling by crosslinking the inhibitory Feγ receptor FcγRIIb with the receptor (Non-Patent Documents 4 and 5).
The first technology of producing bispecific antibodies is a production method using a fusion cell of two hybridomas, called quadroma. Theoretically, tetramers composed of two different H chains and two different L chains are produced as a mixture of up to 10 assemblies, of which only one is the desired antibody having bispecific activity. It is difficult to separate the desired bispecific antibodies from by-products that are similar in physicochemical properties (Non-Patent Document 6).
The known methods for reducing the kind of by-products of the bispecific antibody are a method of introducing a substitution of amino acid residues stabilizing a “heterologous assembly” of the different H chains into CH3 domain (Non-Patent Documents 7, 8, and 9, and Patent Document 4), and a method of stabilizing heterologous assembly of H chains by using chimeric CH3 domains of IgG1 antibody and IgA antibody (Patent Document 1).
In this regard, a method of removing the undesirable by-products generated by “homologous assembly” of homologous H chains during expression of the bispecific antibodies in a purification step has been reported, and a purification method using a difference in binding affinity of IgG antibody subclasses for Protein A has also been known.
Lindhofer et al. have disclosed a purification method of bispecific antibodies consisting of rat IgG2b and mouse IgG2a, based on the properties of rat IgG2b binds to Protein A and mouse IgG2a doesn't bind to Protein A. That is, it is known that a heterodimer of rat IgG2b and mouse IgG2a and a homodimer of mouse IgG2a bind to Protein A, and heterodimeric bispecific antibody can be isolated by pH control during elution (Non-Patent Document 10).
Further, Samuel et al. have proposed a method of specifically purifying human IgG type bispecific antibody by using a difference in the affinity for Protein A. It has been also disclosed that of the two different H chains constituting the bispecific antibody, the CH3 domain of one H chain is subjected to modification of amino acid residues of Arg at position 435 and Phe at position 436 of the EU index, thereby reducing the binding affinity for Protein A, and as a result, heterodimeric IgG type bispecific antibody can be separated and purified from three molecules having different Protein A affinities by pH control during elution (Patent Document 2).
A heterodimeric monovalent antibody consisting of a human IgG1 antibody H chain and a fusion protein of a human κ light chain and an Fc molecule of human IgG1 has been reported as one of the antibody molecules prepared by application of a heterodimeric structure (Patent Document 3).
However, it has been known that when this molecule is expressed, homodimers of H chain-H chain are not secreted, but heterodimers of H chain-fusion protein, homodimers of fusion protein, and multimers are secreted into the culture supernatant, and conjugation of a tag sequence to H chain is required in order to obtain the specific heterodimeric monovalent antibody from the secreted proteins, and this tag should be used for purification. For the use of this monovalent antibody molecule as a drug, side effects caused by possible multimers having unexpected biological activities and a cumbersome and expensive affinity purification using a tag sequence are challenging problems.